Thieno[2,3-b]pyridines as Potassium Channel Inhibitors

ABSTRACT

The invention provides compounds of the formula: 
     
       
         
         
             
             
         
       
         
         
           
             wherein
           R1 is aryl, heteroaryl, cycloalkyl or alkyl;   R2 is H, alkyl, nitro, CO 2 R7, CONR5R6 or halo;   R3 and R4 are H, NR5R6, NC(O)R7, halo, trifluoromethyl, alkyl, CONR5R6, CO 2 R7, nitrile or alkoxy;   R5 and R6 may be the same or different and may be H, alkyl, aryl, heteroaryl or cycloalkyl; or R5 and R6 may together form a saturated, unsaturated or partially saturated 4 to 7 member ring, wherein said ring may optionally comprise one or more further heteroatoms selected from N, O or S;   R7 is H or alkyl;   A is H, halo, or a group of formula X-L-Y;   X is O, S or NR8;   R8 is H or alkyl;   L is (CH 2 ) n , where n is 0, 1, 2, 3 or 4; and   Y is aryl, a heterocyclic group, alkyl, alkenyl or cycloalkyl; the products of mono- and di-oxidation of sulphur and/or mono-oxidation of nitrogen moieties in compounds of formula I;   
         
             or a pharmaceutically acceptable salt thereof. 
           
         
       
    
     These compounds find use as inhibitors of potassium ion channels and thus are useful in the treatment of various conditions including arrhythmia and type-2 diabetes mellitus.

BACKGROUND OF THE INVENTION

The present invention relates to thienopyridine compounds which arepotassium channel inhibitors. Pharmaceutical compositions comprising thecompounds and their use in the treatment of arrhythmia, type-2 diabetesmellitus, immunological disorders, including rheumatoid arthritis,type-1 diabetes, inflammatory bowel disorder and demyelinating disorderssuch as multiple sclerosis are also provided.

Ion channels are proteins that span the lipid bilayer of the cellmembrane and provide an aqueous pathway through which specific ions suchas Na⁺, K⁺, Ca²⁺ and Cl⁻ can pass (Herbert, 1998). Potassium channelsrepresent the largest and most diverse sub-group of ion channels andthey play a central role in regulating the membrane potential andcontrolling cellular excitability (Armstrong & Hille, 1998). Potassiumchannels have been categorized into gene families based on their aminoacid sequence and their biophysical properties (for nomenclature seeGutman et al., 2003).

Compounds which modulate potassium channels have multiple therapeuticapplications in several disease areas including cardiovascular,neuronal, auditory, renal, metabolic and cell proliferation (Shieh etal., 2000; Ford et al., 2002). More specifically potassium channels suchas Kv4.3, Kir2.1, hERG, KCNQ1/minK, IKACh, IkAdo, KATP and Kv1.5 areinvolved in the repolarisation phase of the action potential in cardiacmyocytes. These potassium channels subtypes have been associated withcardiovascular diseases and disorders including long QT syndrome,hypertrophy, ventricular fibrillation, and atrial fibrillation, all ofwhich can cause cardiac failure and fatality (Marban, 2002).

The human delayed rectifier voltage gated potassium channel subunit,Kv1.5, is exclusively expressed in atrial myocytes and is believed tooffer therapeutic opportunities for the management of atrialfibrillation for several different reasons (see review of Brendel andPeukert, 2002): (i) There is evidence that Kv1.5 underlies the cardiacultrarapid delayed rectifier (Kv_((ur))) physiological current in humansdue to similar biophysical and pharmacological properties (Wang et al.,1993; and Fedida et al., 1993). This has been supported with antisenseoligonucleotides to Kv1.5 which have been shown to reduce Kv_((ur))amplitude in human atrial myocytes (Feng et al., 1997). (ii)electrophysiological recordings have demonstrated that Kv_((ur)) isselectively expressed in atrial myocytes, and therefore avoids inducingpotentially fatal ventricular arrhythmia through interfering withventricular repolarisation (Amos et al., 1996; Li et al., 1996; andNattel, 2002). (iii) Inhibiting Kv_((ur)) in atrial fibrillation-typehuman atrial myocytes prolonged the action potential duration comparedto normal healthy human atrial myocytes (Courtemanche et al., 1999).(iv) Prolonging the action potential duration by selectively inhibitingKv1.5 could present safer pharmacological interventions for protectingagainst atrial re-entrant arrhythmias such as atrial fibrillation andatrial flutter compared to traditional class III antiarrythmics, byprolonging the atrial refractory period while leaving ventricularrefractoriness unaltered (Nattel et al., 1999, Knobloch et al., 2002;and Wirth et al., 2003). Class III antiarrythmics have been widelyreported as a preferred method for treating cardiac arrhythmias(Colatsky et al., 1990).

Drugs that maintain the sinus rhythm long-term without proarrhythmic orother side effects are highly desirable and not currently available.Traditional and novel class III antiarrythmic potassium channel blockershave been reported to have a mechanism of action by directly modulatingKv1.5 or Kv_((ur)). The known class III antiarrythmics ambasilide (Fenget al., 1997), quinidine (Wang et al., 1995), clofilium (Malayev et al.,1995) and bertosamil (Godreau et al., 2002) have all been reported aspotassium channel blockers of Kv_((ur)) in human atrial myocytes. Thenovel benzopyran derivative, NIP-142, blocks Kv1.5 channels, prolongsthe atrial refractory period and terminates atrial fibrillation andflutter in in vivo canine models (Matsuda et al., 2001), and S9947inhibited Kv1.5 stably expressed in both Xenopus oocytes and Chinesehamster ovary (CHO) cells and Kv_((ur)) in native rat and human cardiacmyocytes (Bachmann et al., 2001). Elsewhere, other novel potassiumchannel modulators which target Kv1.5 or Kv_((ur)) have been describedfor the treatment of cardiac arrhythmias, these include biphenyls(Peukert et al 2003), thiophene carboxylic acid amides (WO0248131),bisaryl derivatives (WO0244137, WO0246162), carbonamide derivatives(WO0100573, WO0125189) anthranillic acid amides (WO2002100825,WO02088073, WO02087568), dihydropyrimidines (WO0140231), cycloalkylaminederivatives (WO2005018635), isoquionolines (WO2005030791), quinolines(WO2005030792), imidazopyrazines (WO205034837), benzopyranols(WO2005037780), isoquinolinones (WO2005046578), cycloakyl derivatives(WO03063797), indane derivatives (WO0146155 WO9804521), tetralinbenzocycloheptane derivatives (WO9937607), thiazolidone andmetathiazanone derivatives (WO9962891), benzamide derivatives(WO0025774), isoquinoline derivatives (WO0224655), pyridazinonederivatives (WO9818475 WO9818476), chroman derivatives (WO9804542),benzopyran derivatives (WO0121610, WO03000675, WO0121609, WO0125224,WO02064581), benzoxazine derivatives (WO0012492), and the novel compoundA1998 purified from Ocean material (Xu & Xu, 2000).

Compounds that are undergoing development for atrial fibrillation haverecently been reviewed (Page and Rodin, 2005).

Furthermore, the related Kv1.3 channel is expressed in both white andbrown adipose tissue, and skeletal muscle (Xu et al., 2004). Inhibitionof the channel potentiates the hypoglycemic action of insulin, throughincreased insulin-stimulated glucose uptake in these tissues. This issupported by in vivo data, showing that Kv1.3 inhibition in mice withtype-2 diabetes mellitus were significantly more sensitive to insulin.There is strong evidence that Kv1.3 inhibition improves peripheralglucose metabolism by facilitating GLUT4 translocation to the plasmamembrane of adipocytes and myocytes (Desir, 2005). Small moleculeinhibitors of Kv1.3 are emerging as potential targets in the managementof type-2 diabetes, through their actions as insulin sensitisers(WO02-100248).

Human T-lymphocytes possess two types of potassium channels: thevoltage-gated potassium Kv1.3 and the Ca²⁺-activated IKCa1 K⁺ channels(Leonard et al., 1992, Wulff et al., 2003a). These channels set theresting membrane potential of T-lymphocytes, playing a crucial role inthe Ca²⁺ signal transduction pathways that lead to activation of thesecells following antigenic stimulation. Disruption of these pathways canattenuate or prevent the response of T-cells to antigenic challengeresulting in immune suppression (Wulff et al., 2004).

The voltage-gated Kv1.3 and the Ca²⁺-activated IKCa1 K⁺ channels areexpressed in T-cells in distinct patterns that accompany theproliferation, maturation and differentiation of these cells. Theimmunomodulatory effects of channel blockers depends on the expressionlevels of Kv1.3 and IKCa1 channels, which change dramatically whenT-cells transition from resting to activated cells, and duringdifferentiation from the naïve to the memory state. Kv1.3 channelsdominate functionally in quiescent cells of all T-cell subtypes (naïve,T_(CM) and T_(EM)). Activation has diametrically opposite effects onchannel expression; as naïve and T_(CM) cells move from resting toproliferating blast cells, they upregulate IKCa1 channels. Consequentlyactivated naïve and T_(CM) cells express ˜500 IKCa1 channels and anapproximately equivalent number of Kv1.3 channels. In contrast,activation of T_(EM) cells enhances Kv1.3 expression without any changein IKCa1 levels. Functional Kv1.3 expression increases dramatically to1500 Kv1.3 channels/cell, and their proliferation is sensitive to Kv1.3blockers (Wulff et al., 2003, Beeton et al., 2003). B-cells also show aswitch in K⁺ channel during differentiation that parallels the changesseen in the T-cell lineage (Wulff et al., 2004). The discovery that themajority of myelin-reactive T-cells in patents with MS are Kv1.3^(high)T_(EM) cells, has raised interest in the therapeutic potential of Kv1.3blockers in autoimmune disorders (Wulff et al., 2003b, O'Connor et al.,2001). Kv1.3 blockers have been shown to ameliorate adoptive EAE inducedby myelin-specific memory T cells (a model for MS) (Beeton et al., 2001)and to prevent inflammatory bone resorption in experimental periodontaldisease caused mainly by memory cells (Valverde et al., 2005). Inaddition, there is increasing evidence implicating late memory cells inthe pathogenesis of type-1 diabetes, rheumatoid arthritis, psoriasis,inflammatory bowel disorder, Crohn's disease, chronic graft rejectionand chronic graft-vs-host disease (Frierich et al., 2000, Yoon et al.,2001, Viglietta et al., 2002, Yamashita et al., 2004). Specific Kv1.3blockers might therefore constitute a new class of memory-specificimmunomodulators (Shah et al., 2003).

Numerous novel small molecule Kv1.3 channel blockers have been reportedfor the management of autoimmune disorders. These include theiminodihydroquinolines WIN173173 and CP339818 (Nguyen et al., 1996), thebenzhydryl piperidine UK-78,282 (Hanson et al. 1999), correolide (Felixet al., 1999), cyclohexyl-substituted benzamide PAC (U.S. Pat. No.0,619,4458, WO0025774), sulfamidebenzamidoindane (U.S. Pat. No.0,608,3986), Khellinone (Baell et al., 2004),dichloropenylpyrazolopyrimidine (WO-00140231) and psoralens (Wulff etal., 1998, Vennekamp et al., 2004, Schmitz et al., 2005).

Thienopyridines have been reported to be useful as antifungal agents,ligand-gated ion-channel modulators, antibacterials and enzymeinhibitors amongst others.

Thienopyridines substituted at the 2- and 3-positions by hydrogen,alkyl, cycloalkyl or aryl groups, at the 4-position by a hydroxyl group,at the 5-position by a carboxy group and by alkyl or aryl substitutentsat the nitrogen of the 1-position have been claimed as potentantibacterial agents structurally related to the nalidixic acids (Giliset al., 1978).

Thienopyridines substituted at the 3-position by a phenyl group, the2-position by a methyl ketone, the 6-position by a phenyl group, the5-position by a nitrile group or ester and at the 4-position by an aminogroup have been claimed as showing antifungal activity against fungi ofthe family Aspergillus and to inhibit mycotoxin production (Abdelrazeket al., 1992).

Thienopyridines substituted at the 2-, 3- and 6-positions by alkyl oraryl groups, at the 5-position by an ester, aldehyde or 3,5-dihydroxyheptenoic acid derivative and at the 4-position by a substituted phenylgroup have been claimed as potent inhibitors of3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) Reductase in vitro andto show marked cholesterol biosynthesis inhibitory activities in vivo(Suzuki et al., 2001).

Thienopyridines with a fused cycloalkyl ring between the 5- and6-positions, and a phenyl group at the 2- and 3-positions have beenshown to possess poor inhibitory activity against human acetylcholineesterase (Marco et al., 2002).

Thienopyridines have been claimed as anticancer agents with inhibitoryaction against the VEGF-2 receptor tyrosine kinase. Claimed compoundsinclude those thienopyridines substituted at the 2-position with alkylor aromatic moieties, unsubstituted at the 3-position and substituted atthe 4-position by an amino group which may be secondary or tertiary andmay be directly bound to an aromatic or heterocyclic moiety such asphenyl, indole or benzothiazole (U.S. Pat. No. 6,492,383 B1, Munchof etal., 2004).

Thieno[2,3-b]pyridines with a substituted aniline at the 4-position anda substituted phenyl group at the 2-position have been shown to havemodest activity against the Src family of receptor tyrosine kinases aspotential anticancer agents. (Boschelli et al, 2004).

Thieno[2,3-b]pyridines with an amino aryl or amino alkyl substituent atthe 4-position, an amino group at the 3-position and a carbamoylsubstituent at the 2-position have been claimed as modulators of HIVparticle formation and Rev-dependant HIV production. (WO2005076861).

Tricyclic 4-amino-5,6,7,8-tetrahydrothieno[2,3-b]quinoline derivativeshave been claimed as agents for inhibiting acetylcholinesterase andblocking K+ channels, which is claimed to be useful for activatinglowered nerve function induced by senile dementia. (JP04134083).

Thienopyridines with a carbonyl group at the 2-position and an arylgroup at the 3-position have been reported as being useful in thetreatment of osteoporosis (JP07076586).

4-Amino-7-hydroxy-2-methyl-5,6,7,8-tetrahydrobenzo[b]thieno[2,3-b]pyridine-3-carboxylicacid, but-2-ynyl ester (SB205384) and other tricyclic analogues has beenshown to modify the GABA-A receptor modulated chloride current in ratcerebellar granule cells (Meadows et al, 1997).

BRIEF SUMMARY OF THE INVENTION

This invention provides compounds that are potassium channel inhibitors.These compounds are particularly useful for inhibiting one or both ofthe potassium channels Kv1.5 (or Kv_((ur))) and Kv1.3. The Kv1.5 channelis a known target for the treatment of cardiac arrhythmia in the atriasuch as atrial fibrillation (Nattel et al., 1999; Wang et al., 1993);while the Kv1.3 channel is a known target for the treatment of diabetesand immunological disorders. This invention is not limited to thetreatment of these disorders, the compounds also being useful to treatother diseases which require potassium channel inhibition (e.g. asdescribed in Shieh et al., 2000; Ford et al., 2002).

DETAILED DESCRIPTION OF THE INVENTION

Thus, in a first aspect, the present invention provides a compound offormula (I).

Wherein

R1 is aryl, heteroaryl, cycloalkyl or alkyl;

R2 is H, alkyl, nitro, CO₂R7, CONR5R6 or halo;

R3, R4 and R5 are H, NR5R6, NC(O)R7, halo, trifluoromethyl, alkyl,CONR5R6, CO₂R7, nitrile or alkoxy;

R5 and R6 may be the same or different, and may be H, alkyl, aryl,heteroaryl or cycloalkyl; or R5 and R6 may together form a saturated,unsaturated or partially saturated 4 to 7 member ring, wherein said ringmay optionally comprise one or more further heteroatoms selected from N,O or S;

R7 is H, or alkyl;

A is H, halo or a group X-L-Y;

X is O, S or NR8;

R8 is H or alkyl;

L is (CH₂)_(n), where n is 0, 1, 2, 3 or 4; and

Y is aryl, a heterocyclic group, alkyl, alkenyl or cycloalkyl;

the products of mono- and di-oxidation of sulphur and/or mono-oxidationof nitrogen moieties in compounds of formula I;

or a pharmaceutically acceptable salt thereof;

As used herein, an alkyl group or moiety is typically a linear orbranched alkyl group or moiety containing from 1 to 6 carbon atoms, suchas a C₁-C₄ alkyl group or moiety, for example methyl, ethyl, n-propyl,i-propyl, butyl, i-butyl and t-butyl. An alkyl group or moiety may beunsubstituted or substituted at any position. Typically, it isunsubstituted or carries one or two substituents. Suitable substituentsinclude halogen, cyano, nitro, NR9R10, alkoxy, hydroxyl, unsubstitutedaryl, unsubstituted heteroaryl, CO₂R7, C(O)NR9R10, NC(O)R7 andSO₂NR9R10.

As used herein, an aryl group is typically a C₆-C₁₀ aryl group such asphenyl or napthyl. A preferred aryl group is phenyl. An aryl group maybe unsubstituted or substituted at any position. Typically, it carries1, 2, 3 or 4 substituents. Suitable substituents include cyano, halogen,nitro, trifluoromethyl, alkyl, alkylthio, alkoxy, NR9R10, CO₂R7,C(O)NR9R10, NC(O)R7 and SO₂NR9R10 and hydroxyl.

As used herein, a heterocyclic group is a heteroaryl group, typically a5- to 10-membered aromatic ring, such as a 5- or 6-membered ring,containing at least one heteroatom selected from O, S and N. Examplesinclude pyridyl, pyridyl-N-oxide, pyrazinyl, pyrazinyl-N-oxide,pyrimidinyl-N-oxide, pyrimidinyl, pyridazinyl, pyridazinyl-N-oxide,furanyl, thienyl, pyrazolidinyl, pyrrolyl and pyrazolyl groups.Preferred heteroaryl groups are furanyl, thienyl and pyridyl. Examplesof polycyclic heterocycles include indolyl, benzofuranyl,benzothiophenyl and benzodioxolyl. Non-aryl heterocyclic groups are alsoincluded, such as tetrahydrofuranyl or pyrrolidinyl. A heterocyclicgroup may be unsubstituted or substituted at any position. Suitablesubstituents include cyano, nitro, halogen, alkyl, alkylthio, alkoxy,NR9R10, CO₂R7, C(O)NR9R10, NC(O)R7 and SO₂NR9R10 and hydroxyl.

R9 and R10 can be the same or different, and may be selected from H,unsubstituted alkyl, unsubstituted aryl, unsubstituted heteroaryl,unsubstituted cycloalkyl, aminoethyl, methylaminoethyl,dimethylaminoethyl, hydroxyethyl, alkoxyethyl, or R9 and R10 maytogether form a saturated, unsaturated or partially saturated 4 to 7member ring.

When R5 and R6 or R9 and R10 together form a saturated, unsaturated orpartially saturated 4 to 7 member ring, the ring may optionally compriseone, two, or three further heteroatoms.

As used herein, alkoxy means C₁₋₃ alkoxy, cycloalkyl means C₃₋₆cycloalkyl and halogen means Cl, F, Br, or I, preferably Cl, F or Br.

Compounds of formula I wherein mono- and di-oxidation of sulphur and/ormono-oxidation of nitrogen moieties in the compounds has taken place arealso provided. In particular compounds of formula I wherein thethieno[2,3-b]pyridine moiety has been oxidized to one of the followingform an embodiment of the invention:

-   Thieno[2,3-b]pyridine-1-oxide;-   Thieno[2,3-b]pyridine-1,1,-dioxide;-   Thieno[2,3-b]pyridine-1,1,7,-trioxide;-   Thieno[2,3-b]pyridine-1,7,-dioxide; and-   Thieno[2,3-b]pyridine-7-oxide.

Preferred compounds of formula I are those wherein R1 is aryl,cycloalkyl or heteroaryl; R2 is H or alkyl; R3 is H, NR5R6, alkoxy oralkyl; X is O or NR8; R8 is H or alkyl; n is 0, 1, 2, 3 or 4 and Y isalkyl, cycloalkyl, aryl or heteroaryl. Particularly preferred compoundsof formula I are those wherein R1 is aryl or heteroaryl, R2 is H oralkyl, R3 is H, NR5R6, alkoxy or alkyl, X is O or NR8, R8 is H, n is 0,1 or 2 and Y is alkyl, cycloalkyl, aryl or heteroaryl.

Preferred compounds include:

-   (3-Phenyl-thieno[2,3-b]pyridin-4-yl)-pyridin-2-ylmethyl-amine,-   2-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-ylamino}-ethanol,-   2-((2-Hydroxy-ethyl)-{3-phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-amino)-ethanol,-   (6-Chloro-3-phenyl-thieno[2,3-b]pyridin-4-yl)-pyridin-2-ylmethyl-amine,-   [3-(4-Fluoro-phenyl)-thieno[2,3-b]pyridin-4-yl]-pyridin-2-ylmethyl-amine,-   2-[{3-(4-Fluoro-phenyl)-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-(2-hydroxy-ethyl)-amino]-ethanol,-   2-[{3-(4-Fluoro-phenyl)-4-[(6-methyl-pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-(2-hydroxy-ethyl)-amino]-ethanol,-   2-{3-(4-Fluoro-phenyl)-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-ylamino}-ethanol,-   2-{3-(4-Fluoro-phenyl)-4-[(6-methyl-pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-ylamino}-ethanol,-   2-{2-Methyl-3-phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-ylamino}-ethanol,-   [6-Chloro-3-(4-fluoro-phenyl)-thieno[2,3-b]pyridin-4-yl]-pyridin-2-ylmethyl-amine,-   [6-Chloro-3-(4-fluoro-phenyl)-thieno[2,3-b]pyridin-4-yl]-(6-methyl-pyridin-2-ylmethyl)-amine,-   [3-(4-Fluoro-phenyl)-thieno[2,3-b]pyridin-4-yl]-(6-methyl-pyridin-2-ylmethyl)-amine,-   2-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-propane-1,3-diol,    and-   (4-Chloro-3-phenyl-thieno[2,3-b]pyridin-6-yl)-pyridin-2-ylmethyl-amine.

In one embodiment of the first aspect of the invention compounds offormula Ia are provided:

wherein:

R1 is aryl, heteroaryl, cycloalkyl or alkyl;

R2 is H, alkyl, nitro, CO₂R7, CONR5R6 or halo;

R3 and R4 are H, NR5R6, NC(O)R7, halo, trifluoromethyl, alkyl, CONR5R6,CO₂R7, nitrile or alkoxy;

R5 and R6 may be the same or different and may be H, alkyl, aryl,heteroaryl or cycloalkyl; or R5 and R6 may together form a saturated,unsaturated or partially saturated 4 to 7 member ring, wherein said ringmay optionally comprise one or more further heteroatoms selected from N,O or S;

R7 is H or alkyl;

X is O, S or NR8;

L is (CH₂)_(n), where n is 1, 2 or 3; and

Y is aryl, a heterocyclic group, alkyl, alkenyl or cycloalkyl;

or a pharmaceutically acceptable salt thereof.

Compounds of formula I

wherein A is X-L-Y and R3 is NR5R6, nitrile or alkoxy may be synthesisedby reaction of compounds of formula II by displacement of the 6-chlorosubstituent with a suitable nucleophilic species. Such a reaction may becarried out with heating or microwave irradiation optionally in thepresence of solvent and a base.

Compounds of formula II may be synthesized by reaction of compounds offormula III with a suitable nucleophile X-L-Y, where X, L and Y are asdefined herein, optionally in the presence of a solvent and a base, andoptionally at elevated temperature or with microwave irradiation.Preferably the solvent is N-methylpyrrolidinone and the base is ahindered nitrogen base such as triethylamine. If a solvent is presentthe reaction is carried out at the reflux temperature of the solvent, orunder sealed conditions and with microwave irradiation at a temperatureof 120-200° C. Also isolable from this reaction is the product ofsubstitution of the 6-chloro substituent.

Compounds of formula III may be synthesized by reaction of a compound offormula IV with a chlorinating reagent such as phenylphosphonicdichloride or phosphorous oxychloride, optionally in the presence of asuitable solvent.

Compounds of formula IV where R4 is H may be synthesized from compoundsof formula V by decarboxylation. This may be performed at elevatedtemperature, optionally in the presence of a solvent, optionally in thepresence of an inorganic base, and optionally with microwaveirradiation. If a solvent is present the reaction is carried out at thereflux temperature of the solvent, or under sealed conditions and withmicrowave irradiation at a temperature of 120-200° C. Preferably thesolvent is water or ethanol or an admixture thereof and the base is aninorganic hydroxide preferably sodium or potassium hydroxide.

Compounds of formula V may be obtained by cyclisation of compounds offormula VI. This may be performed at elevated temperature, preferably inthe presence of a solvent, preferably in the presence of an inorganicbase, and optionally with microwave irradiation. If a solvent is presentthe reaction is carried out at the reflux temperature of the solvent, orunder sealed conditions and with microwave irradiation at a temperatureof 100-150° C. Preferably the solvent is tetrahydrofuran and the base issodium hydride.

Compounds of formula VI may be synthesized from compounds of formula VIIby reaction with diethyl malonate at elevated temperatures or,preferably, with ethyl malonyl chloride in a suitable solvent,preferably dichloromethane, and an organic nitrogen base, preferablytriethylamine.

A compound of formula VII can be prepared by reaction of a compound offormula VIII with powdered sulphur, under basic conditions and in asuitable solvent. Preferably the base is triethylamine and the reactionis carried out at 25 to 65° C. The solvent may be an alcohol, preferablyethanol.

Compounds of formula VIII can be prepared by heating a compound offormula IX with ethylcyanoacetate (NCCH₂CO₂Et) in the presence of anacid and ammonium acetate in a suitable solvent, optionally withazeotropic water removal. Preferably the acid is acetic acid.

Compounds of formula IX are widely available from commercial sources orcan be readily synthesised using standard synthetic organic chemistryprocedures.

It is understood that compounds of formula I wherein R3 or R4 is an acidor ester group may undergo functional group transformation using methodsfamiliar to those skilled in the art. In a preferred instance suchcompounds may undergo amidation by reaction with an alkyl ordialkylamine, or reduction with a reducing agent such asdiisobutylaluminium hydride or lithium aluminium hydride.

In an alternative process, particularly applicable to those compounds offormula I wherein R1 is aryl, R2 is H, alkyl or halo, and R3 and R4 areH, a compound of formula X is reacted with a suitable nucleophile X-L-Y,where X, L and Y are as defined herein. Optionally the reaction may becarried out in the presence of a solvent and a base, and optionally atelevated temperature or with microwave irradiation. Preferably thesolvent (if present) is an alcohol, preferably ethanol, and the base isa hindered nitrogen base such as triethylamine. If a solvent is presentthe reaction is carried out at the reflux temperature of the solvent, orunder sealed conditions and with microwave irradiation at a temperatureof 120-200° C.

A compound of formula X may be obtained from a compound of formula XI byreaction with a chlorinating reagent such as phenylphosphonic dichlorideor phosphorous oxychloride (or a mixture thereof) in a suitable solventor no solvent, and with heating. Preferably the chlorinating reagent isphosphorous oxychloride and the reaction is carried out at refluxtemperature and in the absence of additional solvent.

Compounds of formula XI may be obtained by the cyclisation of compoundsof formula XII at elevated temperature, optionally in the presence of asolvent, and optionally with microwave irradiation. If a solvent ispresent the reaction is carried out at the reflux temperature of thesolvent, or under sealed conditions and with microwave irradiation at atemperature of 120-200° C. Preferably the solvent is diphenyl ether orDowtherm and the reaction is carried out at reflux temperature.

Compounds of formula XII may be obtained from compounds of formula XIIIby reaction with Mander's reagent (the condensation product of2,2-dimethyl-1,3-dioxane-4,6-dione, commonly known as Meldrum's acid,and triethyl orthoformate), at elevated temperature, optionally in thepresence of a solvent at a temperature of 50-100° C.

Compounds of formula XIII may be obtained from compounds of formula VIIby decarboxylation. This may be performed at elevated temperature,optionally in the presence of a solvent, optionally in the presence of abase, and optionally with microwave irradiation. If a solvent is presentthe reaction is carried out at the reflux temperature of the solvent, orunder sealed conditions and with microwave irradiation at a temperatureof 120-200° C. Preferably the solvent is water or ethanol or anadmixture thereof, and the base is an inorganic hydroxide, preferablysodium or potassium hydroxide.

In an alternative synthesis, suitable for compounds of formula I whereinR3 is H, dechlorination of compounds of formula II is carried out.Suitable conditions include the use of zinc powder in acetic acid at atemperature of 80-118° C. This process may also be applied to the sideproducts of reaction of compounds of formula III with nucleophiles,wherein a compound substituted at the 6-position is formed and theremaining 4-chloro substituent removed to provide compounds of formula Iwherein A is H.

Alternatively, compounds of formula I wherein R3 is a substituted alkylgroup, in a preferred instance an acetic acid ester, can be prepared bythe reaction of a compound of formula XIV, where the 4-position of thethienopyridine ring has a suitable leaving group W, by reaction with asuitable nucleophile X-L-Y, where X, L and Y are as defined herein,optionally in the presence of a solvent and a base, and optionally atelevated temperature or with microwave irradiation. Preferably thesolvent (if present) is toluene, and the base is a hindered nitrogenbase such as triethylamine. In a preferred instance the leaving group Wis a halogen, preferably chlorine, or alternatively an alkyl or arylsulfonate. In a more preferred instance the sulfonate is atrifluoromethanesulfonate. It is understood that compounds of formula Iwherein R3 is an acetic acid ester may undergo transformations usingmethods familiar to those skilled in the art. In a preferred instance,compounds of formula I wherein R3 is a 1-hydroxyethyl group can beprepared by reaction of compounds of formula I wherein R3 is an aceticacid ester with a reducing agent such as diisobutylaluminium hydride orlithium aluminium hydride. In another preferred instance compounds offormula I wherein R3 is a 2-propane-1,3-diol may be obtained byalkylation of compounds of formula I wherein R3 is an acetic acid esterwith a dialkyl carbonate or a chloroformate, and reduction of the1,3-diester formed thereby with a reducing agent such asdiisobutylaluminium hydride or lithium aluminium hydride.

Compounds of formula XIV wherein W is an alkyl or aryl sulfonate can beobtained from a compound of formula XV by reaction with sulfonatingagent, such as a sulfonyl chloride or sulfonic anhydride, in a preferredinstance trifluoromethanesulfonic anhydride, in the presence of asolvent and a base, and optionally at elevated temperature or withmicrowave irradiation. Preferably the solvent is dichloromethane, andthe base is a nitrogen base such as pyridine. Compounds of formula XIVwherein W is a halogen, in a preferred instance chlorine, can beobtained from a compound of formula XV by reaction with a chlorinatingreagent such as phenylphosphonic dichloride or phosphorous oxychloride(or a mixture thereof) in a suitable solvent or no solvent, and withheating. Preferably the chlorinating reagent is phosphorous oxychlorideand the reaction is carried out at reflux temperature and in the absenceof additional solvent.

Compounds of formula XV may be obtained from a compound of formula XVIby an intramolecular cyclisation at elevated temperatures. The reactionmay involve Lewis acid catalysis such as aluminium trichloride, ormineral acid catalysis such as polyphosphoric acid, optionally in thepresence of solvent or as a melt. In a more preferred instance thecyclisation may be induced thermally by heating in a suitablehigh-boiling solvent, optionally in a microwave. A preferred solvent isdiphenyl ether and the reaction is carried out at the reflux temperatureof the solvent.

Compounds of formula XVI may be obtained from compounds of formula XIIIby enamine formation with suitably substituted ketones, in a preferredinstance diethylacetone dicarboxylate. This reaction may be carried outin the presence of solvent under acid catalysis with removal of water byazeotropic distillation or molecular sieves.

Compounds of formula I wherein A is a halogen group, in particular achloride substituent, can be isolated as minor products when a compoundof formula III is reacted with a nucleophile X-L-Y. It will beunderstood by those skilled in the art that further manipulation of thechloride substituent in this instance allows the synthesis of thosecompounds wherein A is hydrogen.

Many of the starting materials referred to in the reactions describedabove are available from commercial sources or can be made by methodscited in the literature references. Synthetic methods forthienopyridines may be found in references such as Gewald et al (1979),Munchof et al (2004), Barker et al (1985), Charvát et al (1995) andarticles cited therein. Synthetic methods can also be found in reviews;thiophenes for example can be found in references cited in ComprehensiveHeterocyclic Chemistry, Eds Katritzky, A. R., Rees, C. R., (4), 863-934,and Comprehensive Heterocyclic Chemistry (II), Eds Katritzky, A. R.,Rees, C. W., Scriven, E. F. V., (2). 607-678.

Suitable starting materials include:

Material Reference Supplier Ethyl Malonyl Chloride 16,387-2 Aldrich4-Fluoroacetophenone F-320-7 Aldrich Acetophenone A1 070-1 Aldrich2-(Aminomethyl)pyridine A6,520-4 Aldrich Diethanolamine D8,330-3 Aldrichethanolamine 41,100-0 Aldrich Propiophenone P5,160-5 Aldrich BenzylamineB1,630-5 Aldrich 2,2-Dimethyl-1,3-dioxane-4,6-dione 21,014-5 AldrichTriethyl orthoformate 30,405-0 Aldrich Diethyl-1,3-Acetone Dicarboxylate16,512-3 Aldrich

As discussed herein, the compounds of the invention are useful in thetreatment of various conditions. Thus, in a second aspect, the presentinvention provides a compound of formula I or Ia as defined herein foruse in medicine. In a further aspect the present invention provides apharmaceutical formulation comprising at least one compound of formula Ior Ia as defined hereinand optionally one or more excipients, carriersor diluents.

The compositions of the invention may be presented in unit dose formscontaining a predetermined amount of each active ingredient per dose.Such a unit may be adapted to provide 5-100 mg/day of the compound,preferably either 5-15 mg/day, 10-30 mg/day, 25-50 mg/day 40-80 mg/dayor 60-100 mg/day. For compounds of formula I, doses in the range100-1000 mg/day are provided, preferably either 100-400 mg/day, 300-600mg/day or 500-1000 mg/day. Such doses can be provided in a single doseor as a number of discrete doses. The ultimate dose will depend on thecondition being treated, the route of administration and the age, weightand condition of the patient and will be at the doctor's discretion.

The compositions of the invention may be adapted for administration byany appropriate route, for example by the oral (including buccal orsublingual), rectal, nasal, topical (including buccal, sublingual ortransdermal), vaginal or parenteral (including subcutaneous,intramuscular, intravenous or intradermal) route. Such formulations maybe prepared by any method known in the art of pharmacy, for example bybringing into association the active ingredient with the carrier(s) orexcipient(s).

Pharmaceutical formulations adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

Pharmaceutical formulations adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active ingredient may be delivered from the patch byiontophoresis as generally described in Pharmaceutical Research, 3(6),318 (1986).

Pharmaceutical formulations adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils.

For applications to the eye or other external tissues, for example themouth and skin, the formulations are preferably applied as a topicalointment or cream. When formulated in an ointment, the active ingredientmay be employed with either a paraffinic or a water-miscible ointmentbase. Alternatively, the active ingredient may be formulated in a creamwith an oil-in-water cream base or a water-in-oil base.

Pharmaceutical formulations adapted for topical administration to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical formulations adapted for topical administration in themouth include lozenges, pastilles and mouth washes.

Pharmaceutical formulations adapted for rectal administration may bepresented as suppositories or enemas.

Pharmaceutical formulations adapted for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns which is administered in themanner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid, foradministration as a nasal spray or as nasal drops, include aqueous oroil solutions of the active ingredient.

Pharmaceutical formulations adapted for administration by inhalationinclude fine particle dusts or mists which may be generated by means ofvarious types of metered dose pressurised aerosols, nebulizers orinsufflators.

Pharmaceutical formulations adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations.

Pharmaceutical formulations adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

Preferred unit dosage formulations are those containing a daily dose orsub-dose, as herein above recited, or an appropriate fraction thereof,of an active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations may also include other agentsconventional in the art having regard to the type of formulation inquestion, for example those suitable for oral administration may includeflavouring agents.

The compounds or compositions of the invention can be used to treatconditions which require inhibition of potassium channels, for examplein the treatment of arrythmia. Thus, in further aspects, the presentinvention provides:

(i) A method of treating or preventing a disorder which requirespotassium channel inhibition, eg arrythmia, type-2 diabetes orimmunological disorders, comprising administering to a subject aneffective amount of at least one compound or pharmaceutical compositionof the invention.

(ii) the use of a compound of the invention in the manufacture of amedicament for use in potassium channel inhibition.

In particular, the medicament is for use in the treatment or preventionof arrhythmia, type-2 diabetes and immunological disorders includingrheumatoid arthritis, type-1 diabetes, inflammatory bowel disorder anddemyelinating disorders such as multiple sclerosis.

EXAMPLES

Using the information outlined herein the following compounds can besynthesised which are given by way of example only. The pharmacologicalprofile of compounds of the present invention can readily be assessed bythose skilled in the art using routine experimentation, such asprocedures and techniques illustrated herein and described in detail inFord et al., 2002.

Example 1 2-Cyano-3-phenyl-but-2-enoic Acid Ethyl Ester

A stirred mixture of acetophenone (180 g, 1.5 mol), ethyl cyanoacetate(170 g, 1.3 mol), ammonium acetate (23.1 g), acetic acid (72 g) andtoluene (300 ml) was heated under reflux for 18 hours while water wasremoved from the reaction by azeotropic distillation. The mixture wasallowed to cool to ambient temperature, toluene (100 ml) was added, thenthe mixture was washed with water (3×100 ml). The combined aqueouswashings were shaken with toluene (50 ml), then the combined toluenesolutions were dried over magnesium sulphate, filtered and the solventwas removed in vacuo. The residual oil was distilled under reducedpressure to give 2-cyano-3-phenyl-but-2-enoic acid ethyl ester as an oilwhich was used without further purification.

Examples 2 and 3

The compounds set out below were prepared in the same way as in Example1, using the appropriate starting materials.

Example Compound 2 2-Cyano-3-(4-fluoro-phenyl)-but-2-enoic acid ethylester 3 2-Cyano-3-phenyl-pent-2-enoic acid ethyl ester

Example 4 2-Amino-4-phenyl-thiophene-3-carboxylic Acid Ethyl Ester

2-Cyano-3-phenyl-but-2-enoic acid ethyl ester (513.25 g, 2.3 mol) wasadded at ambient temperature to a vigorously-stirred suspension ofpowdered sulfur (76 g, 2.3 mol) in ethanol (500 ml). Diethylamine (200ml) was added in portions over 20 minutes, during which time thetemperature of the reaction rose to 62° C. The mixture was allowed tocool to 36° C., then it was heated to 50° C. and stirring at thattemperature was continued for 1 hr. After this time, stirring wasdiscontinued, the hot solution was removed by decantation from unreactedsulfur, then it was allowed to cool to ambient temperature. Theresulting solid was collected by filtration, washed with a little coldethanol and dried in vacuo to give2-amino-4-phenylthiophene-3-carboxylic acid ethyl ester as an orangesolid which was used without further purification.

Examples 5 and 6

The compounds set out below were prepared in the same way as in Example4, using the appropriate starting materials.

Example Compound 5 2-Amino-4-(4-fluoro-phenyl)-thiophene-3-carboxylicacid ethyl ester7 6 2-Amino-5-methyl-4-phenyl-thiophene-3-carboxylicacid ethyl ester

Example 72-(2-Ethoxycarbonyl-acetylamino)-4-phenyl-thiophene-3-carboxylic AcidEthyl Ester

2-Amino-4-phenyl-thiophene-3-carboxylic acid ethyl ester (5.0 g, 0.02M)was dissolved in anhydrous dichloromethane (150 ml). Triethylamine (5.56ml, 0.04M) was added and the mixture cooled to 0° C. Ethyl MalonylChloride (3.79 ml, 0.03M) was added over 5 min maintaining thetemperature at 0° C. The reaction was then stirred at room temperaturefor 1 hr. Water (100 ml) was added and the organic layer separated. Theaqueous layer was extracted with a further 100 ml of dichloromethane.The organics were combined, washed with water (2×100 ml) and dried oversodium sulphate. The concentrated residues were columned on silica,eluting with ethylacetate-cyclohexane 5-10% v/v. Pure fractions werecombined and concentrated and the residues triturated with hexane,decanted and dried to give2-(2-ethoxycarbonyl-acetylamino)-4-phenyl-thiophene-3-carboxylic acidethyl ester as a white solid. Yield=6.95 g (96.2%).

Examples 8 and 9

The compounds set out below were prepared in the same way as in Example7, using the appropriate starting materials.

Example Compound 8 2-(2-Ethoxycarbonyl-acetylamino)-4-(4-fluoro-phenyl)-thiophene-3-carboxylic acid ethyl ester 92-(2-Ethoxycarbonyl-acetylamino)-5-methyl-4-phenyl-thiophene-3-carboxylic acid ethyl ester

Example 104-Hydroxy-6-oxo-3-phenyl-6,7-dihydro-thieno[2,3-b]pyridine-5-carboxylicAcid Ethyl Ester

2-(2-Ethoxycarbonyl-acetylamino)-4-phenyl-thiophene-3-carboxylic acidethyl ester (5.0 g, 13.8 mmol) and sodium hydride (1.1 g, 27.7 mmol) inanhydrous THF (120 ml) were refluxed for 6 hr under nitrogen. Oncooling, solvents were removed in vacuo and the residue suspended inwater (100 ml). The mixture was acidified by addition of concentratedhydrochloric acid (5 ml) and stirred for 1 hr. The precipitate wasfiltered and dried, then recrystallised from ethanol to give4-hydroxy-6-oxo-3-phenyl-6,7-dihydro-thieno[2,3-b]pyridine-5-carboxylicacid ethyl ester as a pale yellow solid. Yield=2.59 g (63.3%).

Examples 11 and 12

The compounds set out below were prepared in the same way as in Example10, using the appropriate starting materials.

Example Compound 11 3-(4-Fluoro-phenyl)-4-hydroxy-6-oxo-6,7-dihydro-thieno[2,3-b]pyridine-5-carboxylic acid ethyl ester 124-Hydroxy-2-methyl-6-oxo-3-phenyl-6,7-dihydro-thieno[2,3-b]pyridine-5-carboxylic acid ethyl ester

Example 13 4-Hydroxy-3-phenyl-7H-thieno[2,3-b]pyridin-6-one

4-Hydroxy-6-oxo-3-phenyl-6,7-dihydro-thieno[2,3-b]pyridine-5-carboxylicacid ethyl ester (1.5 g, 4.76 mmol) was refluxed in 2M sodium hydroxide(50 ml) for 5 hr, filtered while still hot, then cooled to 0° C. andacidified to pH 1 by addition of conc. HCl. The resultant whiteprecipitate was filtered and dried to give4-hydroxy-3-phenyl-7H-thieno[2,3-b]pyridin-6-one as a white solid.Yield=1.05 g (90.7%).

Examples 14 and 15

The compounds set out below were prepared in the same way as in Example13, using the appropriate starting materials.

Example Compound 143-(4-Fluoro-phenyl)-4-hydroxy-7H-thieno[2,3-b]pyridin-6-one 154-Hydroxy-2-methyl-3-phenyl-7H-thieno[2,3-b]pyridin-6-one

Example 16 4,6-Dichloro-3-phenyl-thieno[2,3-b]pyridine

A mixture of 4-hydroxy-3-phenyl-7H-thieno[2,3-b]pyridin-6-one (1.05 g,4.32 mmol) in phenyl phosphonic dichloride (20 ml) was heated to 180° C.for 3 hr. On cooling, the reaction was poured into ice and stirred for30 min. The aqueous was extracted with DCM (3×100 ml). The extracts werecombined, washed with water, dried over sodium sulphate andconcentrated. The residue was columned on silica to give4,6-dichloro-3-phenyl-thieno[2,3-b]pyridine as an opaque oil whichslowly crystallised to a pale yellow solid. Yield=0.505 g (42.9%).

Examples 17 and 18

The compounds set out below were prepared in the same way as in Example16, using the appropriate starting materials.

Example Compound 17 4,6-Dichloro-3-(4-fluoro-phenthieno[2,3-b]pyridine18 4,6-Dichloro-2-methyl-3-phenyl-thieno[2,3-b]pyridine

Examples 19 and 20(6-Chloro-3-phenyl-thieno[2,3-b]pyridin-4-yl)-pyridin-2-ylmethyl-amine

A mixture of 4,6-dichloro-3-phenyl-thieno[2,3-b]pyridine (400 mg, 1.43mmol) and 2-aminomethylpyridine (294 μl, 1.86 mmol) in NMP (1 ml) wereheated in the microwave at 200° C. for 1 h. The reaction was dilutedwith water (30 ml) and DCM (30 ml). The DCM layer was separated andwashed with water (6×50 ml), dried (Na₂SO₄), and concentrated. Theresidue was columned on silica, eluting (EtOAc/Hexane 0-100%). The firstisolated fraction gave(6-chloro-3-phenyl-thieno[2,3-b]pyridin-4-yl)-pyridin-2-ylmethyl-amine(19) as colourless crystals. Yield=288 mg (57.3%). The second isolatedfraction gave(4-Chloro-3-phenyl-thieno[2,3-b]pyridin-6-yl)-pyridin-2-ylmethyl-amine(20) as an orange oil. Yield=24 mg (4.7%).

Examples 21 to 23

The compounds set out below were prepared in the same way as in Example19, using the appropriate starting materials.

Example Compound 21[6-Chloro-3-(4-fluoro-phenyl)-thieno[2,3-b]pyridin-4-yl]-pyridin-2-ylmethyl-amine 22[6-Chloro-3-(4-fluoro-phenyl)-thieno[2,3-b]pyridin-4-yl]-(6-methyl-pyridin-2-ylmethyl)-amine 23(6-Chloro-2-methyl-3-phenyl-thieno[2,3-b]pyridin-4-yl)-pyridin-2-ylmethyl-amine

Example 242-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-ylamino}-ethanol

A mixture of(6-chloro-3-phenyl-thieno[2,3-b]pyridin-4-yl)-pyridin-2-ylmethyl-amine(20 mg, 0.57 mmol) and ethanolamine (1 ml) were heated to 200° C. in themicrowave and maintained at this temperature for 90 min. On cooling, thereaction mixture was poured into DCM (50 ml) and washed with water (2×50ml), dried (Na₂SO₄), and concentrated. The residue was purified onsilica (ethyl acetate, 100%) to give2-{3-phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-ylamino}-ethanolas a white solid. Yield=18 mg (83.9%).

Examples 25 to 30

The compounds set out below were prepared in the same way as in Example24, using the appropriate starting materials.

Example Compound 252-((2-Hydroxy-ethyl)-{3-phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-amino)-ethanol 262-[{3-(4-Fluoro-phenyl)-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-(2-hydroxy-ethyl)-amino]-ethanol 272-{3-(4-Fluoro-phenyl)-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-ylamino}-ethanol 282-[{3-(4-Fluoro-phenyl)-4-[(6-methyl-pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-(2-hydroxy-ethyl)- amino]-ethanol 292-{3-(4-Fluoro-phenyl)-4-[(6-methyl-pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-ylamino}-ethanol 302-{2-Methyl-3-phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-ylamino}-ethanol

Example 31 4-Phenyl-thiophen-2-ylamine

2-Amino-4-phenyl-thiophene-3-carboxylic acid ethyl ester (5 g, 20.2mmol) was suspended in 20% potassium hydroxide solution (100 ml) andheated to reflux. Ethanol (100 ml) was added to aid dissolution. Thereaction was stirred overnight and then cooled to room temperature anddiluted with water (250 ml). The precipitated solid was collected byfiltration and dissolved in DCM/ethyl acetate before drying overmagnesium sulfate. The solvent was removed in vacuo to give4-phenyl-thiophen-2-ylamine. Yield 1.05 g (30%).

Example 322,2-Dimethyl-5-[(4-phenyl-thiophen-2-ylamino)-methylene]-[1,3]dioxane-4,6-dione

Meldrum's acid (1.05 g, 7.3 mmol) was added to triethyl orthoformate (50ml) and stirred at 30° C. for 1 hr. The reaction mixture was cooled toroom temperature and 4-phenyl-thiophene-2-yl-amine (1.07 g, 6.1 mmol)was added in portions. The reaction mixture was heated to 85° C. andstirred at this temperature overnight before cooling to roomtemperature. The solvent was removed in vacuo to give a residue whichwas dissolved in DCM and treated with potassium carbonate. Afterstirring for 30 min the mixture was filtered and the solvent removed invacuo to give2,2-dimethyl-5-[(4-phenyl-thiophen-2-ylamino)-methylene]-[1,3]dioxane-4,6-dione.Yield=1.41 g (70%).

Example 33 3-Phenyl-7H-thieno[2,3-b]pyridine-4-one

Diphenyl ether (15 ml) was heated to reflux and2,2-dimethyl-5-[(4-phenyl-thiophen-2-ylamino)-methylene]-[1,3]dioxane-4,6-dione(1.41 g, 4.29 mmol) was added in portions with evolution of gas. Thereaction mixture was kept at reflux for a further 45 min, before coolingto room temperature. Trituration with diisopropyl ether and petroleumether (40-60°) gave 3-phenyl-7H-thieno[2,3-b]pyridine-4-one. Yield=0.7g, (72%).

Example 34 4-Chloro-3-phenyl-thieno[2,3-b]pyridine

3-Phenyl-7H-thieno[2,3-b]pyridine-4-one (0.7 g, 4.29 mmol) was added tophosphorous oxychloride (10 ml) and heated to reflux for 4 hr. Thesolvent was removed to near dryness and the residue dissolved in DCM.The solution was washed with water followed by saturated sodium hydrogencarbonate and dried over magnesium sulfate. The solution was filteredthrough a pad of silica and solvents removed in vacuo to give4-chloro-3-phenyl-thieno[2,3-b]pyridine. Yield=0.261 g (25%).

Example 35 (3-Phenyl-thieno[2,3-b]pyridin-4-yl)-pyridin-2-ylmethyl-amine

4-Chloro-3-phenyl-thieno[2,3-b]pyridine (47 mg, 0.19 mmol) and2-aminomethyl pyridine (0.5 ml, 4.85 mmol) were placed in a 10 ml glasstube. The vessel was sealed with a septum and placed in the microwavecavity. Using microwave irradiation the temperature was ramped from roomtemperature to 150° C. Once 150° C. was reached, the reaction mixturewas held at this temperature for 90 minutes. After cooling to roomtemperature, the temperature was ramped to 200° C. and held at thistemperature for 30 min. After cooling, the reaction mixture was dilutedwith DCM and washed with water. The organic phase was dried overmagnesium sulfate, filtered and concentrated in vacuo. The residue waspurified by preparative HPLC to give(3-phenyl-thieno[2,3-b]pyridin-4-yl)-pyridin-2-ylmethyl-amine. Yield=14mg, 23%.

Example 36[3-(4-Fluoro-phenyl)-thieno[2,3-b]pyridin-4-yl]-pyridin-2-ylmethyl-amine

[6-Chloro-3-(4-fluoro-phenyl)-thieno[2,3-b]pyridin-4-yl]-pyridin-2-ylmethyl-amine(40 mg, 0.1 mmol) was dissolved in acetic acid (3 ml). Zinc powder (71mg, 1 mmol) was added and the mixture refluxed for 6 hr. On cooling, themixture was filtered through celite and the solids washed with furtherportions of acetic acid (3×5 ml). The acetic acid extracts wereneutralized with saturated sodium bicarbonate solution and extractedwith DCM (2×50 ml). The extracts were combined, dried over sodiumsulfate and concentrated. The residue was purified on silica (ethylactetate/hexane 20-40%) to give[3-(4-fluoro-phenyl)-thieno[2,3-b]pyridin-4-yl]-pyridin-2-ylmethyl-amineas a white solid. Yield=5.5 mg (15.2%).

Example 37

The compound set out below was prepared in the same way as in Example36, using the appropriate starting materials.

Example Compound 37[3-(4-Fluoro-phenyl)-thieno[2,3-b]pyridin-4-yl]-(6-methyl-pyridin-2-ylmethyl)-amine

Example 38 (E/Z)-3-(4-Phenyl-thiophen-2-ylamino)-pent-2-enedloic AcidDiethyl Ester

Amino thiophene (1.39 g, 7.94 mmol), p-Toluenesulfonic acid (7.5 mg, 0.4mmol) and diethylacetonedicarboxylate (1.73 ml, 9.53 mmol) were refluxedfor 12 h in chloroform in the presence of 3 Å molecular sieves. Oncooling, the reaction was filtered, treated with activated charcoal,refiltered through a pad of celite and concentrated. The residue wastriturated with petroleum ether (bp. 40-60°) until crystallisation wascomplete to give (E/Z)-3-(4-Phenyl-thiophen-2-ylamino)-pent-2-enedioicacid diethyl ester as a brown powder. Yield=2.4 g (84%, mixture of E andZ isomers).

Example 39 (4-Oxo-3-phenyl-4,7-dihydro-thieno[2,3-b]pyridin-6-yl)-aceticAcid Ethyl Ester

(E/Z)-3-(4-Phenyl-thiophen-2-ylamino)-pent-2-enedioic acid diethyl esterwas added portionwise to refluxing Diphenyl ether (20 ml). Once additionwas complete, the mixture was refluxed for a further 30 min. On cooling,the reaction was diluted with (bp. 40-60°) petroleum ether (100 ml) andstirred vigorously for 1 h. The initially formed red gum slowlysolidified to a fine yellow precipitate. This was filtered, and washedwith boiling (bp. 40-60°) petroleum ether (2×50 ml) and dried undervacuum to give(4-Oxo-3-phenyl-4,7-dihydro-thieno[2,3-b]pyridin-6-yl)-acetic acid ethylester as a yellow powder. Yield=1.89 g, (71%).

Example 40(3-Phenyl-4-trifluoromethanesulfonyloxy-thieno[2,3-b]pyridin-6-yl)-aceticAcid Ethyl Ester

(4-Oxo-3-phenyl-4,7-dihydro-thieno[2,3-b]pyridin-6-yl)-acetic acid ethylester (0.8 g, 2.5 mmol), and pyridine (0.2 ml, 2.5 mmol) were dissolvedin DCM (5 ml) under nitrogen and cooled to 0° C.Trifluoromethanesulfonic anhydride (0.42 ml, 2.5 mmol) was addeddropwise and the reaction stirred for 18 h at room temperature. Solventswere removed in vacuo, and the residue was diluted with water (100 ml)and extracted with DCM (2×50 ml). The extracts were combined, dried overmagnesium sulphate, filtered and concentrated to a yellow oil, whichsolidified on prolonged standing to a waxy yellow solid. This waspurified on silica, eluting (DCM/petroleum ether (bp. 40-60°)), 0-50%)to give(3-Phenyl-4-trifluoromethanesulfonyloxy-thieno[2,3-b]pyridin-6-yl)-aceticacid ethyl ester as a yellow solid. Yield=878 mg, (78%).

Example 41{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-aceticAcid Ethyl Ester

Thienotriflate (Example 40, 0.9 g, 2 mmol), and Hünigs base (0.7 ml, 4mmol) were dissolved in dry toluene (20 ml) under nitrogen.2-Aminomethylpyridine (0.2 ml, 2 mmol) was added and the mixturerefluxed for 72 h. On cooling, the reaction was washed with water (2×50ml), dried over magnesium sulfate, filtered and concentrated. Theresidue was purified on silica, eluting (Ethyl Acetate/petroleum ether(bp. 40-60°)), 0-50%) to give{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-aceticacid ethyl ester as a yellow solid. Yield=186 mg (26%).

Example 422-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-malonicAcid Diethyl Ester

{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-aceticacid ethyl ester (180 mg, 0.45 mmol) was dissolved in dry THF (10 ml)under nitrogen. Diethyl Carbonate (0.27 ml, 2.23 mmol) was added and themixture cooled to 0° C. Sodium Hydride (36 mg, 0.89 mmol) was added andthe mixture stirred at 0° C. for a further 10 min, then at roomtemperature for 20 min, then brought to reflux for 45 min. On cooling,the reaction was diluted with water (100 ml) and extracted with DCM(3×50 ml), the extracts combined, dried over magnesium sulphate andconcentrated. The residue was purified on silica, eluting (EthylAcetate/DCM), 0-20%) to give2-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-malonicacid diethyl ester as a yellow solid. Yield=96 mg (45%).

Example 432-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-ethanol

{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-aceticacid ethyl ester (47 mg, 0.12 mmol) was dissolved in dry THF (10 ml)under nitrogen and cooled to 0° C. Diisobutyl Aluminium Hydride (0.466ml of a 1M solution in hexanes, 0.466 mmol) was added dropwise over 2min and the mixture stirred at 0° C. for 1 h, then stirred at roomtemperature overnight. The reaction was cooled to OC and water (5 ml)was added carefully, followed by 1M Rochelle's salt (5 ml). The reactionwas stirred at room temperature for 15 min, and then extracted with DCM(2×25 ml), the extracts combined, dried over magnesium sulphate, andconcentrated. The residue was purified on silica, eluting (EthylAcetate/Petroleum ether), 50-100%) to give2-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-ethanolas a brown solid. Yield=25.3 mg (60.4%).

Example 442-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-propane-1,3-diol

A solution of2-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-malonicacid diethyl ester (87 mg, 0.18 mmol) in dry THF (10 ml) was cooled to0° C. under nitrogen. Diisobutyl Aluminium Hydride (1.46 ml of a 1Msolution in hexanes, 1.46 mmol) was added dropwise over 2 min and themixture stirred at 0° C. for 1 h, then allowed to reach room temperatureovernight. The reaction was quenched at 0° C. by adition of 1MRochelle's salt (10 ml). The mixture was extracted with DCM (3×20 ml),the extracts combined, washed with brine (50 ml), dried over magnesiumsulphate and concentrated. The residue was purified on silica, eluting(Ethyl Acetate, 100%) to give2-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-propane-1,3-diolas a yellow oil. Yield=22.5 mg, (31.4%).

Example 45

Analytical Data for compounds representative of the above examples areshown in the table below.

Example Compound Name Mass Spectrum (m/z) 72-(2-Ethoxycarbonyl-acetylamino)-4-phenyl- 7.84 min, 360 (ES−, 100%,thiophene-3-carboxylic acid ethyl ester [M − H]) 82-(2-Ethoxycarbonyl-acetylamino)-4-(4-fluoro- 7.80 min, 378 (ES−, 100%,phenyl)-thiophene-3-carboxylic acid ethyl ester [M − H]) 92-(2-Ethoxycarbonyl-acetylamino)-5-methyl-4- 8.06 min, 374, (ES+,phenyl-thiophene-3-carboxylic acid ethyl ester 100%, [M + H]+) 104-Hydroxy-6-oxo-3-phenyl-6,7-dihydro- 6.50 min, 316 (ES+,thieno[2,3-b]pyridine-5-carboxylic acid ethyl 100%, [M + H]) ester 113-(4-Fluoro-phenyl)-4-hydroxy-6-oxo-6,7- 6.51 min, 334 (ES+,dihydro-thieno[2,3-b]pyridine-5-carboxylic acid 100%, [M + H]) ethylester 12 4-Hydroxy-2-methyl-6-oxo-3-phenyl-6,7- 6.86 min, 330 (ES+,dihydro-thieno[2,3-b]pyridine-5-carboxylic acid 100%, [M + H]) ethylester 13 4-Hydroxy-3-phenyl-7H-thieno[2,3-b]pyridin-6- 5.71 min, 244(ES+, one 100%, [M + H]) 14 3-(4-Fluoro-phenyl)-4-hydroxy-7H-thieno[2,3-5.75 min, 262 (ES+, b]pyridin-6-one 100%, [M + H]) 154-Hydroxy-2-methyl-3-phenyl-7H-thieno[2,3- 5.92 min, 258 (ES+,b]pyridin-6-one 100%, [M + H]) 19(6-Chloro-3-phenyl-thieno[2,3-b]pyridin-4-yl)- 8.00 min, 352 (ES+,pyridin-2-ylmethyl-amine 100%, [M + H]) 21[6-Chloro-3-(4-fluoro-phenyl)-thieno[2,3- 8.16 min, 370, (ES+,b]pyridin-4-yl]-pyridin-2-ylmethyl-amine 100%, [M + H]) 22[6-Chloro-3-(4-fluoro-phenyl)-thieno[2,3- 8.34 min, 384, (ES+,b]pyridin-4-yl]-(6-methyl-pyridin-2-ylmethyl)- 100%, [M + H]) amine 23(6-Chloro-2-methyl-3-phenyl-thieno[2,3- 8.17 min, 366 (ES+,b]pyridin-4-yl)-pyridin-2-ylmethyl-amine 100%, [M + H]) 242-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]- 6.50 min, 377, (ES+,thieno[2,3-b]pyridin-6-ylamino}-ethanol 100%, [M + H]) 252-((2-Hydroxy-ethyl)-{3-phenyl-4-[(pyridin-2- 6.45 min, 421, (ES+ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}- 100%, [M + H])amino)-ethanol 26 2-[{3-(4-Fluoro-phenyl)-4-[(pyridin-2- 6.76 min, 439,(ES+ ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-(2- 100%, [M + H])hydroxy-ethyl)-amino]-ethanol 272-{3-(4-Fluoro-phenyl)-4-[(pyridin-2-ylmethyl)- 6.75 min, 395, (ES+,amino]-thieno[2,3-b]pyridin-6-ylamino}-ethanol 100%, [M + H]) 282-[{3-(4-Fluoro-phenyl)-4-[(6-methyl-pyridin-2- 6.87 min, 453, (ES+,ylmethyl)-amino]-thieno[2,3-b]pyridin-6-yl}-(2- 100%, [M + H])hydroxy-ethyl)-amino]-ethanol 292-{3-(4-Fluoro-phenyl)-4-[(6-methyl-pyridin-2- 6.70 min, 409, (ES+,ylmethyl)-amino]-thieno[2,3-b]pyridin-6- 100%, [M + H]) ylamino}-ethanol30 2-{2-Methyl-3-phenyl-4-[(pyridin-2-ylmethyl)- 6.83, 391 (ES+, 100%,amino]-thieno[2,3-b]pyridin-6-ylamino}-ethanol [M + H]) 36[3-(4-Fluoro-phenyl)-thieno[2,3-b]pyridin-4-yl]- 7.28 min, 336 (ES+,pyridin-2-ylmethyl-amine 100%, [M + H]) 37[3-(4-Fluoro-phenyl)-thieno[2,3-b]pyridin-4-yl]- 7.25 min, 350 (ES+,(6-methyl-pyridin-2-ylmethyl)-amine 100%, [M + H]) 40(3-Phenyl-4-trifluoromethanesulfonyloxy- 7.96 min, 446 (ES+,thieno[2,3-b]pyridin-6-yl)-acetic acid ethyl ester 100%, [M + H]) 41{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]- 7.19 min, 404 (ES+, 100%,thieno[2,3-b]pyridin-6-yl}-acetic acid ethyl ester [M + H]) 422-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]- 7.59 min, 476 (ES+,thieno[2,3-b]pyridin-6-yl}-malonic acid diethyl 100%, [M + H]) ester 432-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]- 6.35 min, 362 (ES+,thieno[2,3-b]pyridin-6-yl}-ethanol 100%, [M + H]) 442-{3-Phenyl-4-[(pyridin-2-ylmethyl)-amino]- 6.09 min, 392 (ES+,thieno[2,3-b]pyridin-6-yl}-propane-1,3-diol 100%, [M + H])

Example 46 Kv1.3 Autopatch Electrophysiology Method

Cells stably transfected with cDNA for human Kv1.3 were grown inDulbecco's Modified Eagle media (DMEM) alpha supplemented with 10% FetalCalf Serum (FCS), 20 μl/ml penicillin (5000 U/ml) streptomycin (5000μg/ml), 10 μl/ml [100×] glutamine, and blasticidin (7.5 μg/ml).Compounds were tested on these cells using the AutoPatch technology inwhole cell mode.

The external bathing solution contained (in mM): 150 NaCl, 10 KCl,1MgCl₂, 3 CaCl₂, 10 HEPES, pH 7.4 with NaOH. Patch pipettes were filledwith an electrode solution of composition (in mM): 100 K-Gluconate, 20KCl, 1MgCl₂, 1 CaCl₂, 10 HEPES, 11 EGTA, 5 ATP-Na₂, 2 Glutathione pH 7.2with KOH.

Compounds were dissolved in DMSO (100%) and made up in the externalbather at a concentration of 1 μM. All experiments were conducted atroom temperature (22-24° C.).

A cell suspension (10 ml), with a density of 100,000 cells/ml, wasaliquoted into a 15 ml centrifuge tube and transferred to an incubator(37° C., 5% CO₂) for approximately one hour before use. Following 60 minincubation, a tube was taken and centrifuged at 1000 rpm for 4 mins atroom temperature. 9.5 ml supernatant was thence discarded, leaving acell pellet at the bottom of the tube. The pellet was then resuspendedusing 100 μl of cold (4° C.), filtered (0.22 μm), 0.2% BSA/bathersolution (0.02 g BSA/10 ml bather). The bottom of the tube was manuallyagitated gently until the solution became cloudy with cells. The 100 μlcell resuspension solution was then stored on the bench at 4° C. (usinga Peltier-based temperature control device) until used.

A length of capillary glass (1B150F-4, WPI) was dipped into the cellsuspension solution, such that ˜3 cm column of fluid was taken up bycapillary action. A Ag/AgCl wire was dropped into the non-dipped end ofthe capillary also. The outside of the solution-filled end of thecapillary was then dried and the capillary was loaded into theAutoPatch™. Borosilicate glass patch pipettes (from 1.5 mm OD,thin-walled filamented, GC150-TF capillary glass, Harvard) were pulledusing a DMZ pipette puller (Zeitz Instruments), and were back-filledusing the internal pipette solution, being careful that no bubblesremain at the tip or in the body of the pipette. Patch pipettestypically had resistances of 2.3-3.5 MΩ. Once filled, the pipette tipand a proportion of the shaft (˜15 mm) were dipped into Sigmacote(Sigma). The recording pipette was then loaded into the AutoPatch™.Automated patch-clamping was initiated by the operator, but thereafterAutoPatch.exe continued the experiment providing that pre-set conditionsand criteria were satisfied.

Whole cell patch-clamp recordings were made using the AutoPatch™ rig,which incorporated an EPC9 or EPC10 amplifier (HEKA, Germany) undercontrol of Pulse software (v8.54 or v8.76, HEKA, Germany), a motioncontroller with 2 translators (Newport, UK), valve controller (VF1) anda c-level suction device all at room temperature (22-24° C.). Thisequipment was completely under the control of AutoPatch.exe and operatorintervention was only made when there was a requirement to refill thedrug reservoirs or to prevent the loss of a cell due to a technicalerror. Cells with an R_(series) greater than 18 MΩ were discounted fromthe experiment.

Qualification stages prior to perfusion and drug application ensuredthat the observed current met the criteria for the experiment. Onlythose cells with an I_(K)>400 pA were used for experiments. Cells werecontinuously perfused with external solution at a flow rate of 1.8-2ml/minute. The perfusion chamber had a working volume of 80-85 μl andallowed for rapid exchange of drug solutions. Online analysis of thehK_(v)1.3 current during the application of compounds was performed bythe AutoPatch™ software. Voltage-step protocols and analysis of data wasperformed as described for conventional electrophysiology.

Electrophysiology voltage-step protocols and analysis of data wasperformed as follows. Data was sampled at 5 kHz, and filtered with a −3dB bandwidth of 2.5 kHz. Cells were held at a voltage of −80 mV.Currents were evoked by a voltage step to +30 mV for 500 ms in durationevery 10 s. Currents were analysed using Pulsefit software (v8.54 orv8.76, HEKA, Germany), with the total charge measured during the wholeof voltage step. All other plots were produced using Igor Pro(WaveMetrics).

Example 47 Kv1.3 Conventional Whole Cell Patch Electrophysiology Method

Cells stably transfected with cDNA for human Kv1.3 were grown inDulbecco's Modified Eagle media (DMEM) alpha supplemented with 10% FetalCalf Serum (FCS), 20 μl/ml penicillin (5000 U/ml) streptomycin (5000μg/ml), 10 μl/ml [100×] glutamine, and blasticidin (7.5 μg/ml).Compounds were tested on these cells using conventionalelectrophysiology equipment in whole cell mode.

The external bathing solution contained (in mM): 150 NaCl, 10Kcl,1MgCl₂, 3CaCl₂, 10 HEPES, pH 7.4 with NaOH. Patch pipettes were filledwith an electrode solution of composition (in mM): 100K-Gluconate,20KCl, 1MgCl₂, 1CaCl₂, 10 HEPES, 11EGTA, 5 ATP-Na₂, 2 Glutathione pH 7.2with KOH.

Compounds were dissolved in DMSO (100%) and made up in the externalbather at a concentration of 1 μM. All experiments were conducted atroom temperature (22-24° C.).

Cells were seeded onto 35 mm plastic culture dishes at varying densitiesand allowed to adhere for at least 4 h before use. Borosilicate glasspatch pipettes (from 1.5 mm OD, thin-walled filamented, GC150-TFcapillary glass, Harvard) were pulled using a Narishege two stagepipette puller and backed filled with internal solution. Patch pipettestypically had resistances of 3.5-4.5 MΩ.

Whole cell patch-clamp recordings were made using a conventional patchclamp rig, which incorporated an EPC9 or EPC10 amplifier (HEKA, Germany)under control of Pulse software (v8.54 or v8.76, HEKA, Germany. Cellswere patched manually and after obtaining the whole cell configuration,drug delivery and experimental parameters were controlled via theAutoPatch™ software. Cells with an R_(series) greater than 18 MΩ werediscounted from the experiment.

Qualification stages prior to drug application ensured that the observedcurrent met the criteria for the experiment. Only those cells with anI_(K)>400 pA were used for experiments. Cells were continuously perfusedwith external solution at a flow rate of 0.5 ml/minute. Online analysisof the hK_(v)1.3 current during the application of compounds wasperformed by the AutoPatch™ software. Voltage-step protocols andanalysis of data was performed using pulsefit (HEKA, Germany).

Electrophysiology voltage-step protocols and analysis of data wasperformed as follows. Data was sampled at 5 kHz, and filtered with a −3dB bandwidth of 2.5 kHz. Cells were held at a voltage of −80 mV.Currents were evoked by a voltage step to +30 mV for 500 ms in durationevery 10 s. Currents were analysed using Pulsefit software (v8.54 orv8.76, HEKA, Germany), with the total charge measured during the wholeof voltage step. All other plots were produced using Igor Pro(WaveMetrics).

Example 48 Kv1.5 Autopatch Electrophysiology Method

Cells stably transfected with cDNA for human Kv1.5 (inpEF6::VA-His-TOPO) were grown in Dulbecco's Modified Eagle media (DMEM)alpha supplemented with 10% Fetal Calf Serum (FCS), 20 μl/ml penicillin(5000 U/ml) streptomycin (5000 μg/ml), 10 μl/ml [100×] glutamine, andblasticidin (7.5 μg/ml). Compounds were tested on these cells using theAutoPatch technology in whole cell mode.

The external bathing solution contained (in mM): 150 NaCl, 10KCl,1MgCl₂, 3CaCl₂, 10 HEPES, pH 7.4 with NaOH. Patch pipettes were filledwith an electrode solution of composition (in mM): 160KCl, 0.5MgCl₂, 10HEPES, 1 EGTA, pH 7.4 with KOH.

Compounds were dissolved in DMSO (100%) and made up in the externalbather at a concentration of 1 μM. All experiments were conducted atroom temperature (22-24° C.).

A cell suspension (10 ml), with a density of 100,000 cells/ml, wasaliquoted into a 15 ml centrifuge tube and transferred to an incubator(37° C., 5% CO₂) for approximately one hour before use. Following 60 minincubation, a tube was taken and centrifuged at 1000 rpm for 4 mins atroom temperature. 9.5 ml supernatant was thence discarded, leaving acell pellet at the bottom of the tube. The pellet was then resuspendedusing 100 μl of cold (4° C.), filtered (0.22 μm), 0.2% BSA/bathersolution (0.02 g BSA/10 ml bather). The bottom of the tube was manuallyagitated gently until the solution became cloudy with cells. The 100 μlcell resuspension solution was then stored on the bench at 4° C. (usinga Peltier-based temperature control device) until used.

A length of capillary glass (1B150F-4, WPI) was dipped into the cellsuspension solution, such that ˜3 cm column of fluid was taken up bycapillary action. A Ag/AgCl wire was dropped into the non-dipped end ofthe capillary also. The outside of the solution-filled end of thecapillary was then dried and the capillary was loaded into theAutoPatch™. Borosilicate glass patch pipettes (from 1.5 mm OD,thin-walled filamented, GC150-TF capillary glass, Harvard) were pulledusing a DMZ pipette puller (Zeitz Instruments), and were back-filledusing the internal pipette solution, being careful that no bubblesremain at the tip or in the body of the pipette. Patch pipettestypically had resistances of 2.3-3.5 MΩ. Once filled, the pipette tipand a proportion of the shaft (a 15 mm) were dipped into Sigmacote(Sigma). The recording pipette was then loaded into the AutoPatch™.Automated patch-clamping was initiated by the operator, but thereafterAutoPatch.exe continued the experiment providing that pre-set conditionsand criteria were satisfied.

Whole cell patch-clamp recordings were made using the AutoPatch™ rig,which incorporated an EPC9 or EPC10 amplifier (HEKA, Germany) undercontrol of Pulse software (v8.54 or v8.76, HEKA, Germany), a motioncontroller with 2 translators (Newport, UK), valve controller (VF1) anda c-level suction device all at room temperature (22-24° C.). Thisequipment was completely under the control of AutoPatch.exe and operatorintervention was only made when there was a requirement to refill thedrug reservoirs or to prevent the loss of a cell due to a technicalerror. Cells with an R_(series) greater than 18 MΩ were discounted fromthe experiment.

Qualification stages prior to perfusion and drug application ensuredthat the observed current met the criteria for the experiment. Onlythose cells with an I_(K)>500 pA were used for experiments. Cells werecontinuously perfused with external solution at a flow rate of 1.8-2ml/minute. The perfusion chamber had a working volume of 80-85 μl andallowed for rapid exchange of drug solutions. Online analysis of thehK_(v)1.5 current during the application of compounds was performed bythe AutoPatch™ software. Voltage-step protocols and analysis of data wasperformed as described for conventional electrophysiology.

Electrophysiology voltage-step protocols and analysis of data wasperformed as follows. Data was sampled at 5 kHz, and filtered with a −3dB bandwidth of 2.5 kHz. Cells were held at a voltage of −80 mV.Currents were evoked by a voltage step to 0 mV for 1000 ms in durationfollowed by a step to ˜0 mV for 1000 ms every 5 s. Currents wereanalysed using Pulsefit software (v8.54 or v8.76, HEKA, Germany), withthe total charge measured during 75-95% of the 0 mV voltage step. Allother plots were produced using Igor Pro (WaveMetrics).

Example 49 Kv1.5 Conventional Whole Cell Patch Electrophysiology Method

Cells stably transfected with cDNA for human Kv1.3 were grown inDulbecco's Modified Eagle media (DMEM) alpha supplemented with 10% FetalCalf Serum (FCS), 20 μl/ml penicillin (5000 U/ml) streptomycin (5000μg/ml), 10 μl/ml [100×] glutamine, and blasticidin (7.5 μg/ml).Compounds were tested on these cells using conventionalelectrophysiology equipment in whole cell mode.

The external bathing solution contained (in mM): 150 NaCl, 10KCl,1MgCl₂, 3CaCl₂, 10 HEPES, pH 7.4 with NaOH. Patch pipettes were filledwith an electrode solution of composition (in mM): 160KCl, 0.5MgCl₂, 10HEPES, 1 EGTA, pH 7.4 with KOH.

Compounds were dissolved in DMSO (100%) and made up in the externalbather at a concentration of 1 μM. All experiments were conducted atroom temperature (22-24° C.).

Cells were seeded onto 35 mm plastic culture dishes at varying densitiesand allowed to adhere for at least 4 h before use. Borosilicate glasspatch pipettes (from 1.5 mm OD, thin-walled filamented, GC150-TFcapillary glass, Harvard) were pulled using a Narishege two stagepipette puller and backed filled with internal solution. Patch pipettestypically had resistances of 3.5-4.5 MΩ.

Whole cell patch-clamp recordings were made using a conventional patchclamp rig, which incorporated an EPC9 or EPC10 amplifier (HEKA, Germany)under control of Pulse software (v8.54 or v8.76, HEKA, Germany. Cellswere patched manually and after obtaining the whole cell configuration,drug delivery and experimental parameters were controlled via theAutoPatch™ software. Cells with an R_(series) greater than 18 MΩ werediscounted from the experiment.

Qualification stages prior to drug application ensured that the observedcurrent met the criteria for the experiment. Only those cells with anI_(K)>400 pA were used for experiments. Cells were continuously perfusedwith external solution at a flow rate of 0.5 ml/minute. Online analysisof the hK_(v)1.3 current during the application of compounds wasperformed by the AutoPatch™ software. Voltage-step protocols andanalysis of data was performed using pulsefit (HEKA, Germany).

Electrophysiology voltage-step protocols and analysis of data wasperformed as follows. Data was sampled at 5 kHz, and filtered with a −3dB bandwidth of 2.5 kHz. Cells were held at a voltage of −80 mV.Currents were evoked by a voltage step to 0 mV for 1000 ms in durationfollowed by a step to ˜0 mV for 1000 ms every 5 s. Currents wereanalysed using Pulsefit software (v8.54 or v8.76, HEKA, Germany), withthe total charge measured during 75-95% of the 0 mV voltage step. Allother plots were produced using Igor Pro (WaveMetrics).

Example 50

Representative biological data is presented below:

Kv1.3 Kv1.5 % Inhibition % Inhibition Example at 1 μM at 1 μM 24 93 7925 82 81 19 72 88 20 99 93 36 32 20 26 45 91 28 20 21 27 72 55 29 62 3121 71 59 22 78 95 37 30 36 35 45 62 43 20 94 44 8 82

Abbreviations

Kv_((ur)) Cardiac Ultrarapid Delayed Rectifier

CHO Chinese Hamster Ovary Cells

DMEM Dulbecco's Modified Eagle media

FCS Fetal Calf Serum

EBSS Earls Balanced Salt Solution

WCPC Whole-Cell Patch-Clamp

DCM Dichloromethane

NMP N-methylpyrrolidinone

HCL Hydrochloric acid

Na₂SO₄ Sodium Sulphate

DME 1,2-Dimethoxyethane

EAE Experimental autoimmune encephalomyelitis

EBSS Earls Balanced Salt Solution

EtOAc Ethyl acetate

EtOH Ethanol

GLUT4 Insulin-regulated glucose transporter

HT Hydroxytryptamine

MgSO4 Magnesium sulfate

MS Multiple sclerosis

T_(CM) Central memory T cell

T_(EM) Effector memory T cell

REFERENCES

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Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed with awide and equivalent range of conditions, formulations, and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents and publications cited herein are fullyincorporated by reference herein in their entireties.

1-11. (canceled)
 12. A method for the prevention or treatment of adisorder which requires potassium channel inhibition, comprisingadministering to a subject an effective amount of at least one compoundof formula I

wherein R1 is aryl, heteroaryl, cycloalkyl or alkyl; R2 is H, alkylnitro, CO₅R7, CONR5R6 or halo; R3 and R4 are H, NR5R6, NC(O)R7, halotrifluoromethyl, alkyl CONR5R6, CO₂R7, nitrile or alkoxy; R5 and R6 maybe the same or different and may be H, alkyl, aryl, heteroaryl orcycloalkyl; or R5 and R6 may together form a saturated, unsaturated orpartially saturated 4 to 7 member rings wherein said ring may optionallycomprise one or more further heteroatoms selected from N, O or S; R7 isH or alkyl; A is H, halo, or a group of formula X-L-Y; X is O, S or NR8;R8 is H or alkyl; L is (CH₂)_(n), where n is 0, 1, 2, 3 or 4; and Y isaryl, a heterocyclic group, alkyl alkenyl or cycloalkyl; or a product ofmono- or di-oxidation of the thieno[2,3-b]pyridine sulphur moiety ormono-oxidation of the thieno[2,3-b]pyridine nitrogen moiety, orcombination thereof; or a pharmaceutically acceptable salt thereof. 13.The method as claimed in claim 12, wherein the disorder is arrhythmia.14. The method as claimed in claim 12, wherein the disorder is type-2diabetes mellitus, an immunological disorder type-1 diabetes,inflammatory bowel disorder or a demyelinating disorder.
 15. The methodof claim 12, wherein said compound of formula I is the product of amono- or di-oxidation of the thieno[2,3-b]pyridine sulphur moiety or amono-oxidation of the thieno[2,3-b]pyridine nitrogen moiety, orcombinations thereof, selected from the group consisting ofThieno[2,3-b]pyridine-1-oxide; Thieno[2,3-b]pyridine-1,1,-dioxide;Thieno[2,3-b]pyridine-1,1,7,-trioxide;Thieno[2,3-b]pyridine-1,7,-dioxide; and Thieno[2,3-b]pyridine-7-oxide.16. The method of claim 12, wherein R1 is aryl or heteroaryl; R2 is H oralkyl, R3 is H, NR5R6, alkoxy or alkyl; X is NR8; R8 is H or methyl; nis 0, 1 or 2 and Y is aryl or heteroaryl.
 17. A method for theprevention or treatment of a disorder which requires potassium channelinhibition, comprising administering to a subject an effective amount ofat least one compound of formula Ia

wherein: R1 is aryl heteroaryl cycloalkyl or alkyl; R2 is H, alkyl,nitro, COR7, CONR5R6 or halo; R3 and R4 are H, NR5R6, NC(O)R7, halo,trifluoromethyl, alkyl, CONR5R6, CO₂R7, nitrile or alkoxy; R5 and R6 maybe the same or different and may be H, alkyl, aryl, heteroaryl orcycloalkyl; or R5 and R6 may together form a saturated, unsaturated orpartially saturated 4 to 7 member ring, wherein said ring may optionallycomprise one or more further heteroatoms selected from N, O or S; R7 isH or alkyl; X is O, S or NR8; L is (CH₂)_(n), where n is 1, 2 or 3; andY is aryl, a heterocyclic group, alkyl, alkenyl or cycloalkyl; or apharmaceutically acceptable salt thereof.
 18. The method as claimed inclaim 17, wherein the disorder is arrhythmia.
 19. The method as claimedin claim 17, wherein the disorder is type-2 diabetes mellitus, animmunological disorder type-1 diabetes, inflammatory bowel disorder or ademyelinating disorder.
 20. (canceled)
 21. The method as claimed inclaim 13, wherein said arrhythmia is atrial fibrillation.
 22. The methodas claimed in claim 14, wherein said immunological disorder isrheumatoid arthritis and said demyelinating disorder is multiplesclerosis.
 23. The method as claimed in claim 18, wherein saidarrhythmia is atrial fibrillation.
 24. The method as claimed in claim19, wherein said immunological disorder is rheumatoid arthritis and saiddemyelinating disorder is multiple sclerosis.